The present invention relates to an enzymatic process for producing cytidine diphosphate choline (hereinafter referred to as CDP-choline) which is useful as a medicine.
CDP-choline is a biosynthetic intermediate for phosphatidyl choline (lecithin), which is a phospholipid, and is useful for the treatment of head injuries, disturbance of consciousness following cerebral surgery, Parkinson""s disease, postapoplectic hemiplegia, etc.
Two kinds of processes are known for producing CDP-choline: chemical synthetic processes disclosed in Japanese Published Examined Patent Applications Nos. 6541/64, 1384/67, 6558/88, etc.; and enzymatic processes utilizing cells of microorganisms such as yeast disclosed in Japanese Published Examined Patent Applications Nos. 2358/73, 40757/73 and 40758/73 and Japanese Published Unexamined Patent Applications Nos. 109996/78, 14593/79 and 313594/88.
A feature common to these known processes is the use of cytosine nucleotides such as cytidine-5xe2x80x2-monophosphate (hereinafter referred to as CMP), cytidine-5xe2x80x2-diphosphate (hereinafter referred to as CDP), cytidine-5xe2x80x2-triphosphate (hereinafter referred to as CTP), and cytosine, or their precursors as starting materials.
Of these starting materials, the basic starting material, CMP, is mainly produced by the RNA (ribonucleic acid) decomposition method which provides four types of nucleotides at the same time. This method is inefficient in that it is impossible to selectively obtain CMP.
The present invention provides a process for producing CDP-choline, which comprises carrying out an enzymatic reaction using cultures of microorganisms having enzyme activities responsible for the production of CDP-choline from orotic acid and choline and/or phosphorylcholine or treatment products of the cultures as the enzyme sources and orotic acid and choline and/or phosphorylcholine as the substrates, allowing CDP-choline to accumulate in the reaction mixture, and recovering CDP-choline from said reaction mixture.
According to the present invention, CDP-choline can be produced with a high efficiency by enzymatic treatment from orotic acid which is easily available from industrial sources, not from a cytosine nucleotide or its precursor.
Orotic acid is a precursor of pyrimidine nucleotides which is used as a hepatotonic. An industrially applicable process is known for producing orotic acid by fermentation of microorganisms belonging to the genus Corynebacterium (EP-A-0312912). As a result of studies to develop a process for producing CDP-choline using orotic acid as a starting material, it has been found that CDP-choline can be produced in a high yield by enzymatic reaction in which a microorganism carrying a recombinant DNA prepared by incorporating into a vector DNA a DNA fragment containing genes coding for CTP synthetase (hereinafter referred to as pyrG), cholinephosphate cytidylyltransferase (hereinafter referred to as CCT) and choline kinase (hereinafter referred to as CKI) and a microorganism which has a high activity of producing uridine-5xe2x80x2-triphosphate (hereinafter referred to as UTP) from orotic acid are used in combination as enzyme sources, and orotic acid and choline and/or phosphorylcholine are used as substrates.